Sodium butyrate regulates phenotypic polarization of BV2 microglia through TLR4/NF-κB signaling pathway

Abstract

Objective To explore the regulatory role and molecular mechanism of sodium butyrate (NaB) in lipopolysaccharide (LPS) induced inflammatory phenotype in BV2 microglia cells and the molecular mechanism. Methods BV2 cells were treated with different concentrations of NaB alone or combined with LPS.CCK8 assay was used to detect the change of cell viability and to screen the optimal concentration of the drug.Then the BV2 cells were divided into control, LPS group, low-and high-dose NaB groups (0.125 and 0.25 mmol/L).An inflammatory model of BV2 cells were established by pretreatment with NaB for 17 h followed by LPS treatment for 24 h.Griess assay was used to measure the NO content in the supernatant, and ELISA was employed to determine the contents of IL-1β, TNF-α and IL-10 in culture medium.The mRNA levels of iNOS and CD206 were detected by RT-qPCR.Immunofluorescence staining was used to observe the expression of iNOS and CD206.The protein levels of TLR4, NF-κB p65m, IKB-α and p-IKB-α were detected by Western blotting.Cytoplasmic and nuclear proteins were isolated to detect the nuclear translocation of NF-κB p65. Results Griess assay showed that the secretion of NO in the high-and low-dose NaB groups were lower than that in the LPS group (P < 0.05).ELISA results indicated that the high-and low-dose NaB groups had lower secretion levels of IL-1β and TNF-α, but high secretion level of IL-10 than the LPS model group (all P < 0.05).RT-qPCR revealed that high-and low-dose NaB treatment significantly decreased the mRNA expression of iNOS (M1 phenotypic marker) and promoted that of CD206(M2 phenotypic marker)(both P < 0.05).Immunofluorescence staining presented similar results as RT-qPCR (P < 0.05).Western blot results displayed that high-and low-dose NaB resulted in significantly inhibited protein expression of TLR4, NF-κB p65 and p-IKB-α as well as nuclear translocation of NF-κB p65 when compared with the LPS group (P < 0.05). Conclusion NaB significantly reduces LPS-induced neuroinflammatory responses in BV2 cells, which might be associated with its inhibition of TLR4/NF-κB signaling pathway and promotion of BV2 cells shifting from M1 to M2 phenotype.

Keywords

• sodium butyrate
• microglia
• inflammation
• polarization
• tlr4/nf-κb signaling pathway